PROJECT SUMMARY ? Additional Tool Development or Data Generation The 3D organization of chromosomes is central to gene regulation and to all aspects of chromosome dynamics. Recent 3C and Hi-C analyses have provided unprecedented detail about the 3D arrangement of chromosomes, and the Center will extend these analyses to provide 4D information. However, there are limitations to 3C/HiC data: first, it provides relative rather than absolute proximity information for gene loci. Second, 3C-based methods tend to have low signal-to-noise ratios, in part due to confounding non-specific contributions of sequence-based propinquity between genetically linked loci. Third, 3C by itself does not provide information about what other molecules (especially proteins and RNAs) are near genetic loci. Therefore, it is essential to develop new tools that provide additional 4D nucleosome organization data sets to both validate and complete the information provided by locus-juxtaposition maps. To do this we will introduce three new tools aimed at providing additional nucleome-mapping data. The first (Aim 1) will be pre- fragmentation of genomes followed by 3C analysis, which we expect to suppress non-specific contacts while maintaining long-range specific contacts. In addition to providing improvements in 3C signal:noise ratios, this approach promises to provide a cost-effective way to generate catalogs of specific looping interactions throughout the genome. The second approach (Aim 2) will be to develop the means to map nuclear components in a completely 3C-orthogonal manner by exploiting the RNA-guided genome binding capacity of nuclease-dead Cas9 (dCas9). In particular, we will adapt dCas9 for spatially restricted proximity labeling, allowing the biotinylation of both proteins and RNAs near specific types of nucleome structural elements. This has the potential to yield spatial position information as well as local protein/RNA enrichment near specific loci, providing insight into the factors that drive or respond to dynamic genome structure. Finally, we will develop new tools for direct, 3C-independent, microscopic visualization and physical study of chromatin in both its mitotic (Aim 3) and interphase (Aim 4) forms. Using micropipette-based microsurgery and microdissection, we will extract metaphase chromosomes and nuclei for combined enzymatic/mechanical study, using Cas9-fusion- protein technology to provide fluorescent labeling of specific chromatin loci. Using multiple labels we will directly test locus-juxtaposition data from Hi-C experiments, and we will be able to study the way that mechanical perturbation of chromosomes and genomes affects relative locus positioning to provide a test of the theoretical models developed in Component 3 of the overall proposal. Collectively, the novel tools that we develop and apply in this component will advance the field of nucleomics by connecting genome structure to the associated proteome and transcriptome, providing unique data on the mechanics of chromosomes and nuclei, and an initial glimpse of how the results of Hi-C and single-cell data correlate with each other.